Paolo Giannetti,1-2 Marios Politis,1 Paul Su,1 Federico Turkheimer,1 Kit Wu,1 Richard Reynolds,1 Richard Nicholas2 and Paola Piccini1

1Centre for Neuroscience and MRC Clinical Sciences Centre, Division of Experimental Medicine, Department of Medicine, Imperial College London, United Kingdom;2Multiple Sclerosis Trials Collaboration, Division of Experimental Medicine, Department of Medicine, Imperial College London, United Kingdom.

ENS Meeting 2011

Multiple Sclerosis Trials Collaboration11L14 Laboratory BlockImperial College LondonCharing Cross CampusLondon W6 8RFTel: +44 0208 383 0675Mobile: +44 07530992684Email: p.giannetti@imperial.ac.uk

Microglia✓Microglia is the first line for central nervous system homeostasis (against any kind of injury)✓Microglia is essential for the onset of progressive neurodegeneration

Microglia can assume different phenotypes according to the microenvironment in which it resides:1. When induced by LPS, IFN-γ or amyloid-β aggregates, microglia acquire features such as Phagocytosis, release of inflammatory mediators and do not support cell renewal, which can even be impeded.2. When induced by IL-4, microglia acquire features such asRemove apoptotic cells, remove myelin debris, release anti-inflammatory factors, support oligodendrogenesis, support neurogenesis, offer neuroprotection.

PK-PET ImagingPositron emission tomography (PET) ligand [(11)C](R)-PK11195 (PK) has high affinity for the translocator protein (TSPO), which is highly expressed in mitochondria of activated microglia.This ligand has been used in several neurological conditions as marker of inflammation and degeneration, as well in MS to identify white and grey matter abnormalities beyond anatomical lesions shown at MRI.

BACKGROUND RESULTS

Relapsing ProgressivePKBPND - EDSS NS Sc = 0.818*, 1

PKBPND - BH lesion load Pc = -0.781*, 2 NSPKBPND - BH volume Sc = -0.101**, 3 NSEDSS - BH lesion load NS NS

* p<0.01** p<0.05Sc: Spearman’s coefficientPc: Pearson coefficient

Table 2: results

Table 1: Study population

Black Holes in Multiple SclerosisDEFINITION✓Black holes (BHs) are multiple sclerosis (MS) lesions appearing hypointense at T1-weighted and hyperintense in T2-weighted magnetic resonance imaging (MRI)

BH PATHOLOGY1. BHs were associated with axonal reduction, extracellular edema and demyelination in 6 CNS bioptised demyelinating lesions (Bruck et al., 1997)

2. In 19 post-mortem MS lesions BHs did not correlate with degree of demyelination or reactive astrocytes but inversely correlated with axonal density (van Walderveen et al., 1998)

3. BHs did not correlate with axonal loss in 14 early MS biopsies. At the MRI follow up the higher the axonal loss and demyelination in the initial biopsy, the more hypointense the lesion became; remyelination was associated with reduction of hypointensity; whether a lesion becomes remyelinated or not, it seems to take place immediately following active demyelination (Bitsch et al., 2001)

4. In EAE, BHs hypointensity correlated with increased cellular infiltrates and reduced myelin content in the lesion centre, with microglial and astroglial infiltrates in the perilesional area (Nessler et al., 2007)

5. Using N-Acetyl-Aspartate spectroscopy, axonal damage was found in severely hypointense T1 lesions (Van Walderveen et al., 1999)

➡ 19 subjects studied ➡ A total of 1 242 BH were detected➡ 947 were found to have a PET detectable PK BPND (Fig. 1),510 pertained to relapsing and 437 to progressive group

✓The results reported refer to the BH with presence of PK binding

10 Relapsing

9 Progressive19 PATIENTS

1 242 Black Holes947 PK

enhancing

295 Not enhancing⧽

⇓t ➚t

➙t

Subject Age Disease duration

EDSS Course

1 26.1 7.7 7.0 Progressive

2 28.9 3.8 7.0 Progressive

3 42.2 19.0 4.0 Relapsing

4 29.4 3.7 9.5 Progressive

5 33.1 7.0 8.0 Relapsing

6 43.6 24.6 3.5 Relapsing

7 47.1 19.2 6.5 Relapsing

8 49.4 31.4 8.0 Progressive

9 57.8 11.3 6.5 Progressive

10 46.5 11.6 6.0 Relapsing

11 42.0 28.1 7.0 Progressive

12 29.6 10.0 7.0 Progressive

13 46.2 25.1 8.5 Progressive

14 43.4 23.0 5.5 Progressive

15 47.6 13.2 4.0 Relapsing

16 39.0 13.3 6.5 Relapsing

17 31.6 13.0 4.0 Relapsing

18 25.1 3.4 4.5 Relapsing

19 26.7 1.0 5.5 Relapsing

Mean (SD) 38.7 (9.6) 14.2 (9.1) 6.2 (1.7) Total

Mean (SD) 38.3 (8.5) 12.5 (7.3) 5.3 (1.5) 10 Relapsing

Mean (SD) 39.2 (11.1) 16.0 (10.9) 7.3 (1.2) 9 Progressive

BH AND DISABILITY• BHs have been associated with irreversible disability

(Truyen et al., 1996; Paolillo et al., 1999; van Walderveen et al., 1999b; Simon et al., 2000)

• but this is not a consistent finding(Giugni et al., 1997; O'Riordan et al., 1998; Masek et al., 2008)

Current imaging techniques have a limited ability to measure the complete spectrum of pathology in MS; BH lesions represent one of the most evident example.

Supported by a2009 EFNSScientific Fellowship

Figure 1 Left: two black holes as seen at MRI.Right: the same black holes show different PK binding at PET scan: 1) absence of tracer uptake; 2) high PK binding.

1: Figure 22: Figure 33: Figure 4

AIMSIn light of the variable relationship of MRI T1 BHs with disability and the variable pathological findings we aim to examine the in-vivo functional microglial activity of T1 BHs and the possible correlation with disability.Microglia activation is here expressed by the PK binding potential of the specifically bound radioligand relative to the non-displaceable radioligand in T1 black holes.

Figure 3Figure 2

SUBJECTSSubjects enrolled (Tab.1)• 10 relapsing MS• 9 progressive MSThe following investigations were performed• History and EDSS• PET [(11)C](R)-PK11195 scan• Colocalizing gadolinium enhancing MRI scan

Figure 4

DISCUSSIONThe results of this study show that relapsing and progressive patients have heterogeneous patterns of 11C-PK11195 uptake in T1 black holes.In progressive subjects PK uptake in BHs was associated with disability suggesting a role for microglia in mediating axonal loss.In relapsing patients PK uptake in BHs was not associated with disability and there was an inverse correlation between PK uptake and black holes lesion load indicating an early inflammatory role for activated microglia.

It is possible that a microglia phenotype switch occurs during the course of MS.Our data could represent the in vivo imaging of two alternative microglia phenotypes in our MS populations.In particular at the lesion onset during an acute inflammatory state a microglia phenotype aiming to recover the lesion seems to be predominant, while in later stages in a chronically over-activated microenvironment, another microglia phenotype is predominant and contributes to the tissue damage.

Figure 2 Correlation between EDSS and PK binding potential in BHsin progressive MS patientsSc = 0.818; p < 0.01

Figure 3 Correlation between BH lesion load and PK binding potentialin relapsing MS patientsPc = - 0.781; p < 0.01

Figure 4 Correlation between BH volume and PK binding potentialin relapsing MS patientsSc = - 0.818; p < 0.05

BLACK HOLES MICROGLIA ACTIVATIONIN RELAPSING AND PROGRESSIVE MULTIPLE SCLEROSIS:

A PET STUDY